The present invention, in some embodiments thereof, relates to methods and kits for ascertaining biosafety of an agent.
Toxicity testing is currently one of the major hurdles for many drug discovery processes. Currently, the main methods for identifying teratogens are either through epidemiological studies in human populations or by controlled exposure of animal models. Specifically, in order to test chemicals for developmental toxicity, OECD (Organization for Economic Co-operation and Development) guidelines require approximately 20 pregnant animals for the control group and each test group, in a minimum of three doses. Following gestation pregnant animals are sacrificed and the litter examined thoroughly for abnormalities [1]. Thus, in vivo screening for teratogenic potential requires the use of a large number of laboratory animals and is very labor intensive and costly. Moreover, there are significant disparities between humans and animal models in their response to chemical substances. Notably, thalidomide is a strong human teratogen that causes many severe congenital defects but was not identified prospectively as such in the mouse [2]. On the other hand, aspirin and caffeine are teratogenic in rodents but not in humans [3,4]. For many of the most prominent teratogens known, the in vivo ‘no observed adverse effect level’ (NOAEL) for mouse and rat, are more than two orders of magnitude higher than the lowest reported teratogenic dose in human. Thus, inferring teratogenicity from in vivo systems (other than primates) is problematic [2]. When a “safe” margin from the animal NOAEL is often considered to be 1/100 or 1/1000 it is difficult in many cases to separate between actual teratogenic effects of compounds and non-specific effects, such as maternal toxicity. In this study we examined the potential of differentiating human embryonic stem cells (HESCs) and induced-pluripotent stem cells (HiPSCs) as an in vitro model for teratogen screening in a human developmental setting, using physiologically relevant doses.
HESCs are cells derived from blastocyst stage embryos and have the unique traits of virtually limitless propagation in culture while retaining their inherent potential to differentiate into cells from the three embryonic germ layers and extraembryonic tissue. When detached from the dish, HESCs aggregate to form embryoid bodies (EBs). They thus begin a process of spontaneous differentiation that closely mimics early human embryogenesis [5,6]. Previous studies using mouse [7-9] and human [10-12] ES cells have mainly focused on cytotoxicity assays to determine developmental toxicity. Mehta et al [11] used, in addition to the cytotoxicity test, also a test for the determination of 12 genetic markers (three for each of: ectoderm, mesoderm endoderm and un-differentiated cells) in order to evaluate the potential of the tested hESCs to differentiate.
U.S. Application No. 20090220996 discloses an embryotoxicity test used in order to replace pre clinical toxicity studies. While this application mainly discloses viability testing in differentiated, partially differentiated and non differentiated cells, it also mentions testing of expression of specific linage markers for the ectoderm, endoderm and mesoderm.
U.S. Application No. 20070248947 discloses testing the toxicity and teratogenicity of compounds on non-differentiated embryonic stem cells by following the metabolome, and testing protein changes in metabolic pathways.